Influenza viruses are enveloped RNA viruses that belong to the family of Orthomyxoviridae (Palese and Shaw, 2007). Influenza A and B viruses are considered to be major human pathogens and in a normal season they can cause between 3-5 million cases of severe illness and up to 500,000 deaths worldwide (World Health Organization, 2003). Influenza A viruses can also cause pandemics such as those that occurred in 1918, 1957 and 1968. These outbreaks resulted in high mortality rates because of the lack of pre-existing immunity against the new virus strain. Since the emergence of the highly pathogenic avian H5N1 influenza virus in the late 1990s (Claas et al., 1998), there have been concerns that it may be the next pandemic virus, which has sparked renewed interest in the development of anti-influenza virus drugs.
Currently there are only four U.S. Food and Drug Administration (FDA)-approved drugs available for the treatment and prevention of influenza. The adamantanes (amantadine and rimantadine) block the M2 ion channel of the virus and prevent the release of the viral genome into the host cell (Pinto and Lamb, 1995; Wharton et al., 1994). These drugs are effective if used prophylactically and if administered within 48 hours of infection but are not effective against influenza B viruses. However, the development of widespread resistance has precluded the use of adamantanes in recent influenza seasons (Bright et al., 2006) and isolates of the H5N1 influenza virus have been shown to be resistant to these drugs due to mutations in M2 (Cheung et al., 2006).
The preferred treatment for influenza virus infection is now the use of the neuraminidase inhibitors, oseltamivir and zanamivir (Garman and Layer, 2004). By targeting the neuraminidase, these compounds prevent the release of the virus from the infected cell and halt the spread of the virus. As part of its pandemic preparedness plan, the World Health Organization (WHO) has advised that supplies of the neuraminidase inhibitors be stockpiled, but it is always advantageous to have at least two antiviral drugs (aimed at different targets) available due to the possible emergence of resistant virus strains. In fact the 2007-2008 influenza season in the Northern hemisphere has shown a marked increase in the number of H1N1 isolates that are resistant to oseltamivir (World Health Organization, 2008) and concerns have also been raised regarding oseltamivir-resistant H5N1 influenza viruses isolated from patients in Southeast Asia (Le et al., 2005).
Vaccination is one means of preventing infection or at least minimizing the severity of disease. Based on knowledge of the current circulating influenza virus strains, the WHO makes an annual decision as to which virus strains should be included in the influenza vaccine for the following season. Manufacturers therefore have a relatively short time period in which to generate new vaccine stocks and this, combined with the increase in demand from the population, sometimes leads to shortages. Vaccine viruses are currently grown in embryonated chicken eggs which generally support high levels of virus growth; however the use of eggs has certain limitations. Vaccine production cannot easily be scaled up at short notice, as would be required during a pandemic, due to the reliance on a continuous supply of embryonated eggs. Furthermore, if the pandemic virus is of avian origin it may be lethal in eggs, as occurred during the preparation of an H5N1 vaccine candidate (Takada et al., 1999). An avian virus would likely also affect the poultry industry and the egg supply may dry up completely. In an effort to avoid these problems, vaccine manufacturers are now establishing tissue culture systems for the growth of influenza virus vaccines (Oxford et al., 2005; Romanova et al., 2004; Tree et al., 2001). The major disadvantage is that wild type human influenza virus strains often do not show optimal growth properties in this culture system, resulting in lower vaccine yields.
Thus, there is an urgent need for the development of new antiviral drugs and also for the improvement of tissue culture-based vaccine production, in preparation for future influenza epidemics or pandemics.